This invention relates to flow cytometric applications to be performed on blood cells and specifically relates to methods for determining the competence of leukocyte phagocytic and leukocyte killing ability by utilizing the methods of flow cytometry.
The monitoring of leukocyte function is becoming of increasing clinical importance, particularly with regard to the pathology of autoimmune and arthritic diseases. Other reasons for inquiry into leukocyte abilities pertain to the healing processes such as those encountered in burn patients. See for instance Ransjo et al., "Some Aspects of Neutrophil Granulocyte Function in Burn Patients", Burns, 5:255-259 (1979), printed in Great Britain and given at the Fifth International Congress on Burn Injuries, Stockholm, June 1978. Further reference may be made to an article by Halgren et al., "The Serum Independent Particle Uptake by PMN From Patients With Rheumatoid, Arthritis and Systemic Lupus Erythematosis", Arthritis and Rheumatism, 21:107-113 (1978) and to Hakansson, et al., "Neutrophil Function In Infection Prone Children", Archives of Disease in Childhood, 55:776-781, (1980).
The particular aspects of leukocyte function monitoring which are becoming of increasing clinical importance involve the cell's ability to phagocytose, i.e., the ability to ingest foreign particles, and the leukocyte's killing ability, i.e., the ability to destroy ingested organisms through the application of reactive oxygen. For further information, reference may be made to articles by Stossel et al., "Quantitative Studies of Phagocytosis by PMN Leukocytes: Use of Emulsions to Measure the Initial Rate of Phagocytosis", The Journal of Clinical Investigations, 51:615-624 (1972); Michell et al., "Measurement of Rates of Phagocytosis: The Use of Cellular Monolayers", J. Cell Biol. 40:216-224, 1969; and Arnaout et al., "Alternative Complement Pathway Dependent Ingestion of Fluolite Particles by Human Granulocytes", The J. of Immunology, 127:278-281 (1981).
Active oxygen is the species of oxygen used by phagocytic cells to kill foreign organisms generally by way of oxidative cytotoxic related mechanisms. It has been previously measured by monitoring neutrophil respiratory events for producing active oxygen and detecting the effects on autofluorescence. See: "Neutrophil Activation Monitored by Flow Cytometry: Stimulation by Phorbol Diester is an All-or-None Event", Science, Vol. 215:673-675 (Feb. 5, 1982). Still other microchemical techniques for the cellular spectrophotometric measurement of oxygen uptake have been described by Glick in an article entitled "Microchemical Analytical Techniques of Potential Clinical Interest", in Clinical Chemistry, Vol. 23, No. 8:1465-1471 (1977). Hultborn et al., described yet additional studies on oxygen consumption rate and nitroblue tetrazolium reduction capacity in the absence and presence of phagocytogenic agents. That article was reported in the Scandanavian Journal of Clinical Laboratory Investigations, 23, 297-304 (1973) in an article entitled, "Studies on Leukocyte Function by Measuring Respiration and Nitroblue Tetrazolium Reduction by Simplified Methods".
Other cellular activation and membrane effects have been described using solid monolayer membranes, fluorescent lipid probes and bound fluorescent antibodies in an article entitled, "Specific Antibody Dependent Interactions Between Macrophages and Lipid Haptens in Planar Lipid Monolayers", Proc. Natl. Acad. Sci., Vol. 78, No. 7:4552-4556 (July 1981). Still other references of general interest include Gordon, "Regulation of Hematopoiesis", Vol. 2, Chapter 42, 1970, Appleton - Century Crofts New York; and Halgren et al. "The Serum-Independent Uptake of IgG-Coated Particles by Polymorphonuclear Leukocytes From Uremic Patients on Regular Dialysis Treatment", J. Lab. Clin. Med. 94:277-284 (1979).
Despite an intense interest in the various phagocytic and killing ability characteristics of leukocytes, the conventional methods have failed to provide suitable methods for the convenient assay of both phagocytic and killing ability.
It is an object of the present invention to provide such methods in a convenient assay and to thereby enable one to efficiently measure both the phagocytic and killing ability of leukocytes. It is another object to allow such measurements to be made on selected leukocyte subclasses. It is a further object to provide such assays for use in flow cytometry type instruments thereby permitting the rapid evaluation of large numbers of cells in a clinically convenient environment and acceptable format.
It is yet another object of the present invention to provide methods capable of relating phagocytic ability of leukocytes with the presence or absence of specified cell surface markers or antigens.